Every biochemical process that happens in an eukaryotic cell relies upon a molecular information flow that leads from receptors that inform the cell about its environment all the way to the molecular effectors that determine the appropriate cellular response. A proper information transmission requires a high degree of organization where the molecular players are organized into different cellular compartments so that the specificity of the cellular response can be properly maintained. Breakdown of this organization is the ultimate cause of all human diseases even if the affected molecular pathways differ according to the kind of disease, such as cancer, diabetes or neurodegenerative diseases just to name a few. Research described in this report has focused on the question of how cells organize their internal membranes to provide with the structural framework on which molecular signaling complexes assemble to ensure proper information processing. These cellular processes are often targeted by cellular pathogens such as viruses to force the cells to produce the pathogen instead of performing the cells normal functions. Better understanding of these processes not only can provide new strategies to fight various human diseases but also to intercept the life cycle of cellular pathogens offering an alternative to antimicrobial drugs. During this period we concentrated on the role(s) of phosphatidylinositol 4-phosphate (PI4P) in the organization of the Golgi. The Golgi is a critically important component of all eukaryotic cells that is involved in the sorting and modification of protein and lipid cargoes destined for secretion or for degradation. The Golgi has high concentration of PI4P made by four different PI 4-kinase enzymes (PI4Ks) all of which can be found in the outer surface of the Golgi membranes. The importance of this high PI4P content is only partially understood. PI4P has been recognized as a docking point for numerous proteins, mostly responsible for non-vesicular transport of structural lipids such as cholesterol. In a collaborative work with the group of Dr. Gustavo Egea in Barcelona, we found that one of the important molecular components of the actin cytoskeleton, betaIII-spectrin is recruited to the Golgi by PI4P. Using newly generated antibodies to specific peptide sequences of the human betaIII-spectrin, they showed that the molecule is found in the Golgi complex, where it is enriched in the Golgi and trans-Golgi network. We then used our recently developed drug-inducible enzymatic tool set to acutely deplete the Golgi-associated pool of PI4P and showed that this manipulation displaced betaIII-spectrin from the Golgi. Similarly, overexpression of a pleckstrin homology domain that specifically recognizes PI4P and competes with proteins that bind to the same lipid, also decreased betaIII-spectrin Golgi localization. Importantly, interference with actin dynamics using various toxins failed to affect the localization of betaIII-spectrin to Golgi membranes. The functional importance of the presence of betaIII-spectrin in the Golgi was demonstrated when depletion of betaIII-spectrin using siRNA technology or the microinjection of anti-&#946;III spectrin antibodies into the cytoplasm lead to the fragmentation of the Golgi. These Golgi fragments showed swollen distal Golgi cisternae and vesicular structures. Using a variety of protein transport assays, the Egea group also showed that the endoplasmic reticulum-to-Golgi and post-Golgi protein transports were impaired in betaIII-spectrin-depleted cells, whereas the internalization of the Shiga toxin subunit B to the endoplasmic reticulum was unaffected. These studies showed that betaIII-spectrin constitutes a major skeletal component of distal Golgi compartments, where it is necessary to maintain the structural integrity and secretory activity of the Golgi, and PI4P appears to be highly relevant for coordinating this process. In another, separate set of studies in collaboration with the group of Dr. Vivek Malhotra, also in Barcelona, the role of PI4P in generating membrane curvature in the Golgi membrane was investigated. BAR (Bin/Amphiphysin/Rvs) domains are protein modules that are found in a large number of membrane-associated molecules that are able to bend biological membranes and generate a curvature that is essential for the budding process. The Malhotra group has shown that the BAR domain containing proteins arfaptin1 and arfaptin2 are localized to the trans-Golgi network (TGN) and, by virtue of their ability to sense and/or generate membrane curvature, could play an important role in the biogenesis of transport carriers. They found that arfaptins contain an amphipathic helix (AH) preceding the BAR domain, which is essential for their binding to PI4P-containing liposomes and the TGN of mammalian cells. The binding of arfaptin1, but not arfaptin2, to PI4P was found to be regulated by protein kinase D (PKD) mediated phosphorylation at Ser100 within the AH. It is of importance that PKD also phosphorylates and activates one of the PI4K enzymes, PI4KB in the Golgi that generates most of the PI4P in this compartment. Using our drug-inducible enzymatic tool set to deplete the Golgi-associated pool of PI4P we showed that arfaptins require PI4P to be able to reassociate with the Golgi once they are released by brefeldin A treatment. Notably, only arfaptin1 was found to be required for the PKD-dependent trafficking of chromogranin A by the regulated secretory pathway. Altogether, these findings revealed the importance of PI4P and PKD in the recruitment of arfaptins to the TGN and their requirement in the events leading to the biogenesis of secretory storage granules.